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Structural insights into metazoan pretargeting GET complexes

Nature Structural & Molecular Biology volume 28, pages 1029–1037 (2021)Cite this article

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Abstract

Close coordination between chaperones is essential for protein biosynthesis, including the delivery of tail-anchored (TA) proteins containing a single C-terminal transmembrane domain to the endoplasmic reticulum (ER) by the conserved GET pathway. For successful targeting, nascent TA proteins must be promptly chaperoned and loaded onto the cytosolic ATPase Get3 through a transfer reaction involving the chaperone SGTA and bridging factors Get4, Ubl4a and Bag6. Here, we report cryo-electron microscopy structures of metazoan pretargeting GET complexes at 3.3–3.6 Å. The structures reveal that Get3 helix 8 and the Get4 C terminus form a composite lid over the Get3 substrate-binding chamber that is opened by SGTA. Another interaction with Get4 prevents formation of Get3 helix 4, which links the substrate chamber and ATPase domain. Both interactions facilitate TA protein transfer from SGTA to Get3. Our findings show how the pretargeting complex primes Get3 for coordinated client loading and ER targeting.

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Fig. 1: Cryo-EM structures of pretargeting metazoan GET complexes.
Fig. 2: Get4 primes Get3 for TA protein loading.
Fig. 3: A composite lid covers the Get3 substrate chamber.
Fig. 4: Secondary Get3–Get4 interactions mediate TA protein transfer.
Fig. 5: SGTA remodels one bridging complex and the substrate chamber lid.
Fig. 6: Validation of the metazoan pretargeting GET complex architecture.

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Data availability

EM maps and models described in this study are available under accession nos. EMD-24700, EMD-24701, EMD-24702, EMD-24703, EMD-24704, PDB-7RU9, PDB-7RUA and PDB-7RUC. Source data are provided with this paper.

Code availability

No code was generated in this study.

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Acknowledgements

Cryo-EM screening and data collections were performed at the Cryo-EM Center for Structural Biology and the Molecular Electron Microscopy Suite at Harvard Medical School. Data processing was supported by SBGrid. SEC-MALS was performed at the Center of Macromolecular Interactions at Harvard Medical School. We thank M. McKenna for calmodulin complexes; M. Chambers, Z. Li, S. Sterling and R. Walsh for cryo-EM support; K. Arnett for SEC-MALS training; R. Keenan and C. Atkinson for input at preliminary stages of this project; and A. Brown, R. Hegde and Shao Laboratory members for helpful discussions. This work was supported by the Richard and Susan Smith Family Foundation (S.S.), Harvard Medical School (S.S.), the Vallee Foundation (S.S.), the Packard Foundation (S.S.) and NIH DP2GM137415 (S.S.). M.C.J.Y. is supported by AHA predoctoral fellowship no. 287375208.

Author information

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Blavatnik Institute, Boston, MA, USA

    Alexander F. A. Keszei, Matthew C. J. Yip, Ta-Chien Hsieh & Sichen Shao

  2. Massachusetts College of Pharmacy and Health Sciences, Boston, MA, USA

    Ta-Chien Hsieh

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Contributions

A.F.A.K., M.C.J.Y., T.-C.H. and S.S. performed and analyzed experiments. A.F.A.K. collected cryo-EM data. A.F.A.K. and S.S. processed cryo-EM data, built atomic models and wrote the paper with input from all authors. S.S. supervised the project.

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Correspondence to Sichen Shao.

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Peer review information Nature Structural & Molecular Biology thanks Shu-ou Shan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Florian Ullrich was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 GET complexes analyzed in this study.

a, SDS-PAGE and Coomassie staining of recombinant cBUGG (cBag6-Ubl4a-Get4-Get3) and cBUGGS (cBag6-Ubl4a-Get4-Get3-SGTA) complexes, representative of 6 independent purifications. b, Recombinant Flag-tagged tail-anchored (TA) protein containing the UV-activatable crosslinking amino acid Bpa in the Sec61β transmembrane domain [Flag-TA(Bpa)] in complex with the calcium-dependent chaperone calmodulin was incubated with SGTA, cBUGG, and the calcium chelator EGTA as indicated. Reactions were exposed to UV light and analyzed by SDS-PAGE and immunoblotting, representative of 2 independent experiments. TA protein crosslinks to other TA protein molecules (x TA), SGTA, and Get3 are indicated.

Source data

Extended Data Fig. 2 Quality of maps and models.

a, Representative 2D class averages of the cBUGG (cBag6-Ubl4a-Get4-Get3; top) and cBUGGS (cBUGG + SGTA; bottom) complexes. Scale bars, 100 Å. Pink arrowheads, extra density seen in 2D class averages of cBUGGS but not cBUGG. b, Fourier shell correlation (FSC) coefficient vs. resolution (1/Å) curves of the indicated maps. Resolution was estimated at FSC = 0.143 (gray dotted line). c, Unsharpened Coulomb potential maps colored by local resolution. Get3 L(α4) is indicated. Light orange arrows, C-terminal region of Get4; Pink arrow, cBUGGS-specific interaction. d, Model vs. map FSC curves.

Extended Data Fig. 3 Examples of model and map fits.

Segmented EM densities of the sharpened cBUGG-in map and atomic model of the indicated Get3 (green), Get4 (light orange), and cBag6 (blue) residues. Map contour levels are listed below.

Extended Data Fig. 4 Get3 comparisons.

a, Superposition of Get3 from cBUGG-in (colored) with yeast Get3 in the closed conformation (gray, PDB 2WOJ). The two Get3 subunits are differentiated by dark (Get3-A) and light (Get3-B) green. Helices lining the substrate chamber are labeled. b, Superposition of Get3 from cBUGG-in (green) with yeast Get3 (gray) bound to the Pep12 transmembrane domain (TMD, purple, PDB 4XTR). c, Clipped view of the metazoan Get3 substrate chamber colored by surface hydrophobicity (blue, least hydrophobic to orange, most hydrophobic). d, Segmented EM density (at 14.1σ contour level) and atomic model of the ATP binding site of cBUGG-in. The catalytic Asp68 mutated to Asn is indicated. e, Superposition of the ATP-binding sites of Get3 in cBUGG-out with yeast Get3 in the closed conformation (2WOJ), bound to a substrate (4XTR), and in complex with Get4/5 (4PWX). Resolutions of crystal structures are reported in parentheses. Motifs involved in ATP binding and hydrolysis are labeled; other residues are transparent.

Extended Data Fig. 5 Get3-Get4 comparisons.

a, Superposition of cBUGG-out (colored) with yeast Get3-Get4-Get5 (gray, PDB 4PWX). Conserved (black) and L(α4) (purple) binding interactions are boxed. b, Surface electrostatics of the Get3-Get4 interface. Boxed regions correspond to panel a. c, Superposition of the bridging factors of cBUGG-in (gray) and cBUGG-out (colored) aligned on Get3. d, Overview of Get3-Get4 interactions along one bridging arm. Note that the two interaction sites on Get4 involve different Get3 subunits.

Extended Data Fig. 6 Substrate chamber lid.

a, Lid region above the Get3 substrate chamber of the unsharpened cBUGG map without imposed symmetry (at 9.5σ contour level; gray) and the cBUGG-out atomic model (colored). Dotted lines indicate proposed continuity of unmodeled regions of Get3 (green) and Get4 (yellow). b, Unmodeled sequences of Get3 and Get4. Hydrophobic (orange), acidic (red), and basic (blue) amino acids are colored. Modeled amino acids are gray with Get3 secondary structure designations above (green). Mauve line, Get4 residues modeled in PDB 6AU8; gray dashed lines, unmodeled helices predicted by PSIPRED. c-d, Unsharpened cBUGG-in map (at 9.7σ contour level) and hypothetical lid helices contributed by c, Get3 or d, Get4. Mauve arrow indicates break in lid density that may correspond to the point where Get3 loops back towards α9. e, Cartoon (top) and surface electrostatics (bottom) of Get3 in cBUGG-in showing a basic face of α7 (dashed rectangle) that may interact with acidic C-terminal Get4 residues. Basic residues (Arg183 and Arg179) along α7 are shown. f, Scheme for assaying radiolabeled TA protein capture and transfer from SGTA to Get3. IVT, in vitro translation. g, SDS-PAGE and autoradiography (top) or Coomassie staining (bottom) of PURE in vitro translation reactions with no additional chaperone, SGTA, or the indicated Get3 variant, followed by chemical crosslinking. Note that the KAAKKK Get3 mutant captures TA protein as well as wildtype Get3, representative of 2 independent experiments.

Source data

Extended Data Fig. 7 Recruitment platform and SGTA interactions.

a, Model and unsharpened map of cBUGG-in showing connectivity of cBag6 and cBag6 interactions with Get4. Blue arrow, turning point after cBag6 α3. b, cBUGG-out model of a bridging arm fitted into the unsharpened cBUGG map without imposed symmetry. Red arrow, interaction between Ubl4a and the cBag6 α2-α3 loop; red box, interaction between the C terminus of cBag6 and the Get4 α9-α10 loop. Blue dotted line, map region corresponding to unmodeled C-terminal cBag6 residues. c, Different views of the unsharpened cBUGGS map at 9.5σ contour level as in Fig. 5b. Relevant helices of Get3 (green), Get4 (light orange), and cBag6 (blue) are numbered. Arrows are as in Fig. 5b. d, The masked cBUGGS map (pink) aligned to the cBUGG map (transparent gray), both at 4.5σ contour level, showing remodeling of the recruitment platform towards D1 (blue arrow) and the region above the Get3 substrate chamber upon SGTA binding. The view on the right corresponds to the top view in Fig. 1 (right panels). e, Aligned maps as in panel d, both at 9.5σ contour level, showing how SGTA binding remodels the lid over the Get3 substrate chamber.

Extended Data Fig. 8 Identification of Get3(Bpa) crosslinks to SGTA and Get4.

UV-dependent crosslinking reactions as in Fig. 6b were subjected to denaturing pulldowns for Get3-Strep and immunoblotted for a, SGTA (Ponceau staining shown in bottom panel) or b, FLAG-tagged Get4, representative of 2 independent experiments. Low levels of uncrosslinked Get4 result from non-specific interactions with the resin, are a small proportion of the input (see Fig. 6b), and serve as loading controls.

Source data

Extended Data Fig. 9 Influences on complex architecture.

a, SEC-MALS traces of the indicated complexes. Absorbance at 280 nm was normalized to the highest peak for each sample. *, minor populations of higher-order cBUGGS (black) and excess Get4 (orange). b, SDS-PAGE and Coomassie staining of cBUGGS purified via Flag-tagged Get4, representative of 2 independent purifications. c, Representative 2D classes (top, scale bar, 100 Å) and micrographs (bottom, scale bar, 50 nm) of negatively stained cBUGGS purified via GST- (left) or Flag-tagged Get4 (right). d, SDS-PAGE and Coomassie staining of the BUGGS complex containing full-length Bag6 assembled with Ubl4a, Get4, Get3, and SGTA, representative of 3 independent experiments.

Source data

Supplementary information

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Supplementary Figs. 1–5 and discussion.

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Supplementary Video 1

cBUGG complex overview.

Supplementary Video 2

Comparison between cBUGG-in and cBUGG-out conformations.

Supplementary Video 3

cBUGGs complex overview.

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Keszei, A.F.A., Yip, M.C.J., Hsieh, TC. et al. Structural insights into metazoan pretargeting GET complexes. Nat Struct Mol Biol 28, 1029–1037 (2021). https://doi.org/10.1038/s41594-021-00690-7

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